This document discusses a study on the transient expression of the Red Fluorescent Protein (RFP) gene in maize (Zea mays) callus tissue using particle bombardment. Specifically, it aims to evaluate the tissue culture response of different maize genotypes, construct an expression vector for particle-inflow gun mediated transformation of maize calli, and determine the strength and localization of RFP gene expression using confocal microscopy. Ten maize genotypes were chosen for the study. The expression vector will contain the RFP gene driven by the coconut promoter Cocosin. RFP expression will be observed after calli bombardment with the RFP gene coated on tungsten particles. This study hopes to contribute to genetic transformation methods in

1. TRANSIENT EXPRESSION OF MAIZE (Zea mays) CALLI USING THE
RED FLUORESCENT PROTEIN (RFP) GENE FROM Discosoma sp.
Advances on both callus production and transformation have provided resources for genetic
diversity and useful tools for successful breeding programs. One of the many crops suitable for this is
maize, the country’s secondary staple crop which could have different possible sources of explants like
immature embryos, immature tassels and ears. The manipulation of plant cells, tissues and organs in
vitro is producing an increasing number of unique clones of industrial, biochemical, genotypic and
agronomic importance.
Principal transformation methods, though perfectly established often undergo changes or
improvements to achieve recalcitrant species transformation or higher frequencies. Some plants or
genotypes within a crop exhibit variability on the level of expression of a particular gene being
introduced. The same is true to what is observed in the transformation studies of maize crop. It is for
this reason that the researcher is prompted to study the efficacy of the transient expression of RFP
gene in maize. Specifically, it will seek to (a) evaluate tissue culture response of the maize genotypes
being used, (b) construct expression vector for particle-inflow gun (PIG)-mediated maize calli
transformation; and (c) to determine relative strength of gene expression in terms of intensity and
localization by confocal microscopy.
Evaluation of the tissue culture includes the identification of the types of callus produced using
the dicamba and silver nitrate growth regulators, comparing mean number of the types of callus
observed among the genotypes inoculated and the growth regulators used and determining whether
there is a difference on the frequency of embryogenic calli being produced using the two growth
regulators. Maize genotypes chosen in this study are Var4, CML 482, CML 484, P9, P29, P51, Pi17,
Pi23, CML 161 and CML 451.
The expression vector for PIG-mediated maize calli transformation will use the pBI121. Full
length RFP gene will be from pDsRed2. Cocosin, isolated from coconut will be used as the promoter to
drive the RFP gene. The gene construct will be transformed into the competent cell E.coli strain DH5α.
Selection of the confirmed insertion of the RFP transgene will be through the ability of the cells to
confer kanamycin resistance and those that would appear red on the plates on the LB medium.
The isolated and replicated gene coated with tungsten particles will be bombarded to the
selected calli of maize genotypes. Distance (in) and pressure (kPA) will serve as the two parameters.
Selection will be applied to screen for transgenic cells. Non-transgenic cells will be removed. Gene
expression in terms of intensity and localization will be determined through confocal microscopy.
There have been previous studies on transient expression, only most of it utilized beta-
glucuronidase (GUS) and green fluorescent protein (GFP) genes. This time, the Red Fluorescent Protein
(RFP) gene will be used. The results of the study are hoped to be of wide use of genetic stable
transformation which will be the next wave of the coconut gene expression in a model monocot Zea
mays project and from which this study is anchored.
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Submitted by: Rheomie D. Opiasa MBB 299 20-Oct 2014
Submitted to: Dr. Ma. Genaleen Q. Diaz